Drug Resistant Staphylococci are a Major Medical Problem
Staphylococci (especially S. aureus and S. epidermidis) are major human pathogens and are the most common cause of nosocomial infections reported in the U.S. Each year approximately two million hospitalizations result in nosocomial infections, increasing hospital death rate in the U.S. by 35%. S. aureus and S. epidermidis infections are the leading cause of nosocomial pneumonia, surgical site and bloodstream infections, medical device associated infections, as well as community-acquired infections such as osteomyelitis and septic arthritis, skin infections, endocarditis, and meningitis (Rubin R J et al., Emerg Infect Dis. 1999; 5:9-17 [1]). Currently, more than 95% of patients with staphylococcal infections worldwide do not respond to first-line antibiotics such as penicillin or ampicillin. Drug-resistant staphylococcus was once largely confined to hospitals and nursing homes but is now spreading to communities as well (Lowy F D. N. Engl. J. Med 1998; 339:520-532 [2]). The emergence and spread of drug-resistant bacteria underscores the need to find new modes of prevention and alternative antibiotic treatment to bacterial infections, including S. aureus, S. epidermidis and others. The instant invention addresses this need and others.
S. aureus Cause Diseases by Producing Virulence Factors
S. aureus are part of the normal flora of the human skin, but can cause fatal diseases due to the formation of biofilms and/or the production of toxic exomolecules. The toxins include toxic-shock syndrome toxin-1 (TSST-1), a pyrogenic toxin that causes toxic shock syndrome; Staphylococcal enterotoxins, a major cause of food poisoning; Proteases that allow the bacteria to exploit its environment of metabolites and enable its spread within the host; Hemolysins, leukocidin and other virulence factors that are expressed, secreted or sequestered by staphylococci and have been shown to affect the outcome of the infective process [2].
A novel approach for therapy development is to interfere with staphylococcal virulence (biofilm formation and toxin production). Eliminating the production of virulence factors does not only make the bacteria far less pathogenic, but may also make the bacteria more susceptible to host immune defenses and to conventional antibiotics (Balaban N, et al., Science 1998; 280:438-440 [3]).
Regulation of Staphylococcal Virulence by Quorum Sensing
Biofilm formation and toxin production is regulated by a quorum sensing mechanism, where molecules produced and secreted by the bacteria (autoinducers) reach a threshold concentration and activate signal transduction pathways, leading to activation of the genes that encode for virulence factors [2].
S. aureus express surface molecules such as fibronectin binding-proteins, fibrinogen binding-protein and protein A, in the early exponential phase of growth, when the bacteria are in lower density [2]. Expression of adhesion molecules allows the bacteria to adhere to and colonize host cells and implanted medical devices. When in higher densities, the bacteria produce toxic exomolecules such as Toxic Shock Syndrome Toxin-1 (TSST-1), enterotoxins, proteases and hemolysins that allow the bacteria to survive, disseminate and establish the infection [2].
The ability of the bacteria to express adhesion molecules and colonize when in lower densities and to express toxic exomolecules and cause disease when in higher densities, is due to a complex regulatory process, which involves quorum sensing (QS) mechanisms (Kleerebezem M, et al., Mol. Microbiol. 1997; 24: 895-904 [4]) and activation of genetic loci such as traP (Balaban N et al., J Biol Chem 2001; 276:2658-2667 [5]) agr (Lina G. et al., Mol Microbiol 1998; 28:655-662 [6], sar (Heyer G, et al., Infect. Immun. 2002; 70:127-133 [7]), sae (Giraudo A T et al., FEMS Microbiol. Lett. 1999; 177:15-22 [8]). These processes act in parallel or in concert to regulate virulence [5].
To date, two staphylococcal quorum-sensing systems (SQS) have been described. SQS 1 consists of the autoinducer RNAIII-activating protein (RAP) and its target molecule TRAP [3,5]. RAP is a protein of about 33 kDa [3] that is an ortholog of the ribosomal protein L2 (coded by the rplB gene) and usually consists of 277 amino acids. RplB is highly conserved among eubacteria ((see detailed description below). TRAP is a ˜21 kDa protein that is phosphorylated in the presence of RAP [5]. The sequence of TRAP is highly conserved among staphylococcal strains and species and its secondary structure is highly conserved among gram positive bacteria. TRAP usually consists of 167 amino acids (see detailed description below).
SQS 2 is composed of the products of the gene regulatory system agr. agr encodes two divergently transcribed transcripts, RNAII and RNAIII (Novick R P, et al, Mol. Gen. Genet. 1995; 248:446-458 [9], and Novick R P et al, EMBO J. 1993; 12:3967-3975 [10]). RNAII is a polycistronic transcript that encodes AgrA, AgrC, AgrD and AgrB, where AgrD is a pro-peptide that yields an autoinducing peptide (AIP) that is processed and secreted with the aid of AgrB (Otto M. Peptides 2001; 22:1603-1608 [1] and Saenz H L et al., Arch Microbiol 2000; 174:452-455 [12]). Once agr is activated and AIP is secreted, it induces the phosphorylation of AgrC [6] and AgrA, leading to the production of the regulatory RNA molecule termed RNAIII [10]. RNAIII upregulates the production of numerous secreted toxins [10].
Interaction Between SQS1 and SQS 2
RAP induces the phosphorylation of its target molecule TRAP, leading in a yet unknown manner both to increased cell adhesion and to the activation of agr. Once agr is activated and RNAII is produced (in the midexponential phase of growth), the octapeptide (AIP) and its receptor AgrC are made. AIP downregulates TRAP phosphorylation [5](leading to reduced adhesion properties) and independently to upregulation of the phosphorylation of its receptor, AgrC [6]. This leads to the phosphorylation of AgrA, resulting in the production of RNAIII. RNAIII leads to the expression of many toxic exomolecules [10].
Inhibition of SQS 1: A Novel Mode of Therapy and Prevention of Bacterial Infections
Mice that were vaccinated with RAP (native or recombinant) were protected from a challenge of S. aureus [3]. This confirms the important role of RAP in S. aureus pathogenesis and opens an opportunity for the development of a new vaccine.
Staphylococcal infections can be inhibited by RNAIII inhibiting peptide (RIP). RIP inhibits Staphylococci from adhering and from producing toxins by interfering with the known function of SQS 1. RIP competes with RAP on inducing TRAP phosphorylation, thus leading to inhibition of the phosphorylation of TRAP [5]. This leads to a decrease in cell adhesion and biofilm formation, to inhibition of RNAIII synthesis and to suppression of the virulence phenotype [3]. The peptide RIP was first isolated from culture supernatants of coagulase negative staphylococci that were identified with 99% certainty to be S. xylosus. The sequence of RIP was identified as YSPXTNF, (SEQ ID NO: 22) where X can be a Cys, a Trp, or a modified amino acid, as well as peptide derivatives like YKPITN (SEQ ID NO: 25) (Gov Y, et al., Peptides, 2001; 22:1609-1920 [13]. Synthetic RIP analogues were designed in their amide form as YSPWTNF(—NH2) (SEQ ID NO: 26) and shown to be extremely effective in inhibiting RNAIII in vitro and in suppressing S. aureus infections in vivo, including: cellulitis (tested in mice against S. aureus Smith Diffuse), septic arthritis (tested in mice against S. aureus LS−1), keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), and mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) (Balaban N, et al., Peptides 2000; 21:1301-1311 [14]. These findings indicate that RIP can serve as a useful therapeutic molecule to prevent and suppress staphylococcal infections.
Biofilm-Related Infections
Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections (Costerton J W, et al. Science. 1999; 21:284:1318-1322 [15]). Biofilms develop preferentially on inert surfaces, or on dead tissue, and occur commonly on medical devices and fragments of dead tissue such as sequestra of dead bone; they can also form on living tissues, as in the case of endocarditis. Biofilms grow slowly, in one or more locations, and biofilm infections are often slow to produce overt symptoms. Sessile bacterial cells release antigens and stimulate the production of antibodies, but the antibodies are not effective in killing bacteria within biofilms and may cause immune complex damage to surrounding tissues. Even in individuals with excellent cellular and humoral immune reactions, biofilm infections are rarely resolved by the host defense mechanisms. Antibiotic therapy typically reverses the symptoms caused by planktonic cells released from the biofilm, but fails to kill the biofilm. For this reason biofilm infections typically show recurring symptoms, after cycles of antibiotic therapy, until the sessile population is surgically removed from the body. It is therefore important to prevent biofilm formation rather than to try to eradicate biofilms once they have formed.
As shown in Table 1, many of biofilm-related nocosomial infections are caused by staphylococci [15].
TABLE 1Partial list of human nosocomial infections involving biofilms.Infection or diseaseCommon biofilm bacterial speciesSuturesS. aureus and Staphylococcus epidermidisExit sitesS. aureus and S. epidermidisArteriovenous shuntsS. aureus and S. epidermidisSchleral bucklesGram-positive cocciContact lensP. aeruginosa and Gram-positive cocciUrinary catheter cystitisE. coli and other Gram-negative rodsPeritoneal dialysis (CAPD)Staphylococci, variety of bacteria and fungiperitonitisEndotracheal tubesa variety of bacteria and fungiHickman cathetersS. epidermidis and C. albicansICU pneumoniaGram-negative rodsCentral venous cathetersS. epidermidis and othersMechanical heart valvesS. aureus and S. epidermidisVascular graftsGram-positive cocciOrthopedic devicesS. aureus and S. epidermidisPenile prosthesesS. aureus and S. epidermidis
RIP Reduces Bacterial Adhesion
RIP decreased bacterial adhesion to eukaryotic cells (tested on HEp2 cells) and to plastic (tested on polystyrene, silicone and polyurethane (Balaban N, et al., Kidney Int. 2003; 63:340-345 [16]). RIP could be used to coat medical devices to prevent staphylococcal infections.
RIP Deviates from AIP
RIP deviates from AIP in that RIP is a linear peptide [13] while AIP must contain a thiolactone structure to be active [11], the sensor of RIP is TRAP [5] while the sensor of AIP is AgrC [6], RIP inhibits both cell adhesion and toxin production [16] while inhibitory AIPs inhibit toxin production but activate cell adhesion (Vuong C, Saenz H L, Gotz F, Otto M. Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis 2000; 182:1688-1693 [17].
Molecular Mechanisms of RIP
While the specific molecular mechanisms are not fully understood, it is known that RIP inhibits agr expression (RNAII and RNAIII [13]) and therefore inhibits the production of toxins (Vieira-da-Motta O, et al., Peptides, 2001; 22:1621-1628 [18]). It is known that RIP regulates cell adhesion in an agr-independent mechanism, because adhesion of agr null cells is equally inhibited in the presence of RIP as the wild type [16]. Because RIP inhibits TRAP phosphorylation [5] and TRAP has been demonstrated to be essential for cell adhesion, agr expression and pathogenesis (see detailed description below), RIP regulates S. aureus pathogenesis via TRAP, and perhaps via additional targets.
The mechanism through which RIP inhibits quorum sensing mechanisms, discussed above, involves inhibition of the phosphorylation of TRAP. There is evidence of the presence of TRAP and TRAP phosphorylation in S. epidermidis (see detailed description below), indicating that there is a similar quorum sensing mechanisms both in S. aureus and in S. epidermidis and the potential for RIP to interfere with biofilm formation and infections caused by both species. In addition, there is evidence that TRAP is conserved among all staphylococcal strains and species, and thus that other Staphyloccoal species have a similar quorum sensing mechanism as described above. As a result, RIP should be effective against any type of staphylococcus. 
RAP and TRAP are Target Sites for Therapy in Many Types of Bacteria
Other infection-causing bacteria appear to have proteins with sequence similarity to TRAP. These bacteria include Bacillus subtilus, Bacillus anthracis, Bacillus cereus, Listeria innocua, Listeria monoctogenes (see detailed description of invention).
Still further, RAP is an ortholog of the ribosomal protein L2, encoded by the rplB gene. L2 is highly conserved among bacteria, including specifically Streptococcus ssp, Listeria spp, Lactococcus-spp, Enterococcus spp, Escherichia coli, Clostridium acetobtylicum, and Bacillus spp. This finding indicates that treatment aimed at disturbing the function of RAP in S. aureus will also be effective in treating L2-synthesizing bacteria as well (see detailed description).